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Advanced Cell Signalling > Protein Kinases Respond to Stress > Flashcards

Protein Kinases Respond to Stress Flashcards

1
Q

What was protein kinases being able to respond to stress identified in?

A

Budding yeast (Saccharomyces Cerevisiae)

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2
Q

How does yeast grow if there is plentiful amounts of glucose?

A

Anaerobically by fermentation (they produce ethanol instead of lactate)

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3
Q

What do yeast do if glucose is absent?

A

They induce genes which encode mitochondrial proteins needed for aerobic metabolism
OR
They switch on genes required for growth on other fermentable carbon sources

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4
Q

Give examples of genes required for growth on other fermentable carbon sources

A

SUC2
MAL
GAL

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5
Q

Function of SUC2 genes

A

Encodes invertase

Needed for the breakdown of sucrose to glucose + fructose

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6
Q

Function of MAL genes

A

Needed for the breakdown of maltose to glucose

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7
Q

Function of GAL genes

A

Needed for the fermentation of galactose

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8
Q

What system do yeast use to detect glucose starvation and switch on the genes required for metabolic changes?

A

SNF1 kinase complex

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9
Q

What is sfn4?

A

A regulator of Snf1 that represses its kinase activity when glucose is present

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10
Q

How was sfn4 function discovered?

A

Overexpression of Snf1 could partly overcome a mutation in Snf4, but not vise versa

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11
Q

What is two-hybrid analysis used for?

A

To test if two proteins interact in vivo

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12
Q

Function of DNA binding domain

A

To bind to DNA at the promoter

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13
Q

Function of activator domain

A

To bind to co-activators to recruit RNA polymerase

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14
Q

How does the two-hybrid analysis work?

A

DNA binding domain is bound to protein A
Activator domain is bound to protein B
If A and B interact, the reporter gene will be switched on, otherwise it will not be

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15
Q

What is Gal4?

A

A protein expressed in yeast

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16
Q

What did the two hybrid analysis reveal in terms of Snf1 and Snf4

A

Snf1 bound to the DNA binding domain of Gal4
Snf4 bound to the activator domain of Gal4
Therefore they work as a complex

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17
Q

Name 3 Snf1-interacting proteins

A

Sip1 (Snf1-interacting protein 1)
Sip2
Gal 83

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18
Q

What is Snf1 and Snf4 unable to grow on?

A

Sucrose, or a non-fermentable carbon source, e.g. ethanol

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19
Q

What happens you delete one of Sip1, Sip2 and Gal83

A

nothing

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20
Q

What happens if you delete Sip1, Sip2 and Gal83 together?

A

You produce the same phenotype as deleting Snf4 or Snf1

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21
Q

What 3 seperate complexes does Snf1 exist as?

A

Snf1:Snf4:Sip1
Snf1:Snf4:Sip2
Snf1:Snf4:Gal83

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22
Q

What are the 3 subunits of AMPK? And what is their ratio?

A

alpha
beta
gamma
1:1:1

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23
Q

What is the alpha subunit of AMPK related to?

A

Snf1

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24
Q

What is the beta subunit of AMPK related to?

A

Sip1/Sip2/Gal83

25
Q

What is the gamma subunit of AMPK related to?

A

Snf4

26
Q

What isoforms do the AMPK subunits exist as?

A

alpha1 and alpha 2
beta1 and beta2
gamma1, gamma2 and gamma3

27
Q

How is AMPK activated?

A

Allosterically by AMP

By upstream kinases which phosphorylate it on Thr172 of the activation loop of the kinase domain

28
Q

Where is Thr172 found?

A

In Snf1

29
Q

What happens if Thr172 is mutated?

A

Snf1 function is abolished

30
Q

How many kinases does the yeast genome contain?

A

120

31
Q

How was Snf1 and AMPK upstream kinase identified?

A

A library of the 120 yeast strains were made, each overexpression a dingle protein kinase fused to GST
All of the 120 GST-kinase fusions were purified on glutathione-sepharose
Only Elm1 activated AMPK and Snf1 in cell-free assays
KO of Elm1 did not induce a Snf1 phenotype
Pak1 and Tos3 (closely related protein kinases) identified
KO of all 3 induced a Snf1-like phenotype
Adding any back restored growth

32
Q

What was Pak1 renamed to?

A

Snf1-activating kinase (Sak1)

33
Q

What are the closest relatives of Elm1, Pak1 and Tos3 in the human genome?

A

Liver kinase B1 (LKB1)

Calmodulin-dependent kinase (CaMKK1/2)

34
Q

What does mutations in LKB1 induce?

A 
Peutz-Jeghers Syndrome (inherited susceptibilty to cancer)
Lung cancer (20%)
Cervical cancer (10%)
35
Q

Describe Peutz-Jeghers Syndrome

A

Patients are heterozygotes
Develop frequent benign intestinal tumours and have an increased risk of developing malignant cancers at other sites through loss of their one normal gene copy

36
Q

What 2 accessory subunits are bound to LKB1?

A

STRAD

MO25

37
Q

What does LBK1:STRAD:MO25 induce?

A

Phosphorylation and activation of AMPK

38
Q

Name 2 treatments which cause AMPK activation in cells lacking LKB1

A

AICAR

Phenformin

39
Q

How do treatments induce AMPK activation?

A

Increase AMP levels

40
Q

When is LKB1 active?

A

It is always active

41
Q

What does AMP do to AMPK in terms of LKB1?

A

Makes Thr172 a better substrate for LKB1

42
Q

What does AMP do to AMPK in terms of protein phosphatases?

A

Makes Thr172 a worse substrate for protein phosphatases

43
Q

Can AMP influence AMPK when it is already phosphorylated?

A

It can allosterically activate it

44
Q

Why does AMP:ATP increase effect AMPK activation more than ADP:ATP?

A

Adenylate kinase is activated

This wants to maintain the [ATP][AMP]/[ADP]2 as close to the equilibrium ration of ~1 at all times

45
Q

Is there phosphorylation of AMPK when LKB1 is absent?

A

Yes, a small amount

46
Q

What increases p-AMPK when LKB1 is absent?

A

Adding Ca2+ ionophore A23187

47
Q

When is the function of A23187?

A

Promotes entry of Ca2+ from the extracellular medium, increasing the Ca2+ in the cytoplasm

48
Q

What CaMKK inhibitor inhibited p-AMPK by A23187?

A

STO-609

49
Q

KO of which CaMKK inhibited AMPK activation by A23187?

A

CaMKK2

50
Q

What is the second upstream kinase of AMPK?

A

CaMKK2

51
Q

In cells expressing CaMKK2, what else increases AMPK activation?

A

Hormones which increase cell Ca2+

52
Q

How do hormones induce intracellular Ca2+?

A

They bind to receptors which release intracellular Ca2+

53
Q

What does intracellular Ca2+ induce?

A

A demand for ATP by:

  • triggering energy-requiring processes such as secretion
  • the need to remove Ca2+ back out of the cytosol
54
Q

How does AICAR work?

A

It is a nucleotide which is taken up and converted to AMP

55
Q

How does metformin induce AMPK?

A

Inhibit mitochondrial ATP synthesis and thus increase AMP

56
Q

What is the issue with metformin?

A

They deplete ATP and therefore are non-specific and can have off target effects

57
Q

Name a drug which binds to AMPK distally from the AMP binding domain to promote AMPK activation

A

A-769662

58
Q

Are there any specific AMPK inhibitors and what is used?

A

No

Compound C is used but this targets other kinases too

Advanced Cell Signalling (12 decks)

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